Detection of autoantibodies reactive with pancreatic islet cell antigenic molecules and/or insulin

Abstract

A method of screening a sample of body fluid obtained from an animal subject for analyte autoantibodies reactive with one or more antigenic molecules selected from pancreatic islet cell antigenic molecules and insulin, or one or more variants, analogues, derivatives or fragments thereof, and a kit for use in such a method.

The invention claimed is: 
     
       1. A method of screening a sample of serum obtained from an animal subject for analyte autoantibodies reactive with one or more antigenic molecules selected from GAD 65 , GAD 67 , and IA-2, or one or more variants, analogues, derivatives or fragments thereof, said method comprising:
 (a) providing an undiluted sample of serum from said subject; 
 (b) providing one or more first sources of antigenic molecules with which analyte autoantibodies when present in said sample can interact, wherein the one or more first sources of antigenic molecules are provided in an amount so as to form a monovalent intermediate complex comprising [antigenic molecule]- [analyte autoantibody] capable of forming a divalent complex with antigenic molecules of one or more second sources wherein said divalent complex comprises [antigenic molecule of said first source]- [analyte autoantibody]- [antigenic molecule of said second source]; 
 the one or more first sources of antigenic molecules comprising
 (i) a first source of GAD antigenic molecules selected from GAD 65 , GAD 67 , one or more variants, analogues, derivatives or fragments thereof and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from GAD 65 , GAD 67 , IA-2 and said one or more variants, analogues, derivatives or fragments thereof and wherein at least one of said antigenic molecules of said fusion molecule comprises GAD 65 , GAD 67 , or one or more variants, analogues, derivatives or fragments thereof, and 
 (ii) a first source of IA-2 antigenic molecules selected from IA-2, one or more variants, analogues, derivatives or fragments thereof, and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from IA-2, GAD 65 , GAD 67  and said one or more variants, analogues, derivatives or fragments thereof and wherein at least one of said antigenic molecules of said fusion molecule comprises IA-2 or one or more variants, analogues, derivatives or fragments thereof; 
 
 (c) providing one or more second sources of antigenic molecules with which analyte autoantibodies when present in said sample of serum can interact, wherein the one or more second sources of antigenic molecules comprises
 (i) a second source of GAD antigenic molecules selected from GAD 65 , GAD 67 , one or more variants, analogues, derivatives or fragments thereof and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from GAD 65 , GAD 67 , IA-2 and said one or more variants, analogues, derivatives or fragments thereof and wherein at least one of said antigenic molecules of said fusion molecule comprises GAD 65 , GAD 67 , or one or more variants, analogues, derivatives or fragments thereof, and 
 (ii) a second source of IA-2 antigenic molecules selected from IA-2, one or more variants, analogues, derivatives or fragments thereof, and fusion molecules comprising two or more directly or indirectly fused antigenic molecules selected from IA-2, GAD 65 , GAD 67  and said one or more variants, analogues, derivatives or fragments thereof and wherein at least one of said antigenic molecules of said fusion molecule comprises IA-2 or one or more variants, analogues, derivatives or fragments thereof; 
 
 (d) contacting said antigenic molecules as provided by steps (b) and (c) with said sample of serum being screened, whereby analyte autoantibodies when present in said sample of serum can interact with said antigenic molecules so as to form one or more complexes comprising [antigenic molecule of said first source]- [analyte autoantibody]- [antigenic molecule of said second source], wherein said antigenic molecules of said first and second sources when present in said one or more complexes comprise, or are derived from, a common antigenic molecule, or wherein binding regions of said antigenic molecules of said first and second sources, for said analyte autoantibody, when present in said one or more complexes are present in, or are derived from, a common antigenic molecule; 
 (e) prior to step (d), providing immobilising means whereby said antigenic molecule of said first source as present in a complex as formed in step (d) is immobilized to a solid support prior to step (d) by coating said solid support with said antigenic molecule of said first source; 
 (f) prior to, or concurrent with, or subsequent to, step (d), providing direct or indirect detectable enzymatic label whereby said antigenic molecule of said second source as present in a complex as formed in step (d) is provided with such direct or indirect detectable enzymatic label prior to, or concurrent with, or subsequent to, step (d); and 
 (g) detecting the presence of complexes formed in (d) immobilized according to (e) so as to provide an indication of analyte autoantibodies present in said sample. 
 
     
     
       2. A method according to  claim 1 , wherein the antigenic molecules of the first and second sources when present in said one or more complexes comprise one or more common antigenic molecules. 
     
     
       3. A method according to  claim 2 , wherein antigenic molecules of said first and second sources when present in the one or more complexes comprise either GAD 65  or GAD 67 . 
     
     
       4. A method according to  claim 1 , wherein one of the antigenic molecules of the first or second source when present in the one or more complexes comprises GAD 65  or GAD 67 , the other antigenic molecule when present in the one or more complexes comprises one or more variants, analogues, derivatives or fragments of GAD 65 , GAD 67 , with which analyte autoantibodies when present in said sample of serum can interact. 
     
     
       5. A method according to  claim 1  wherein said undiluted sample of serum is obtained from an animal subject suspected of suffering from, susceptible to or having one or more of the following disease states—type 1 diabetes mellitus and/or stiff man syndrome, type 2 diabetes mellitus, one or more autoimmune thyroid diseases, celiac disease, one or more connective tissue diseases, adrenal autoimmunity, or a combination of two or more different autoimmune diseases. 
     
     
       6. A method according to  claim 1 , wherein said indirect enzymatic label comprises alkaline phosphatase or horse radish peroxidase.